Escherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE ), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyAas shown by primer extension. The corresponding putative promoter contains an unusual −10 sequence (TATGAAT) that is separated from a conventional −35 sequence by a GC-rich spacer. Site-directed deletion of the G in the −10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the G+C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5–3 nm.